Ultrasensitive Multiplexed Immunoassay for Tumor Biomarkers Based on DNA Hybridization Chain Reaction Amplifying Signal

Abstract

In this work, a novel electrochemical immunoassay protocol has been reported for simultaneous determination of multiple tumor biomarkers based on DNA hybridization chain reaction (HCR) for signal amplification. Alpha-fetoprotein (AFP) and prostate specific antigen (PSA) were selected as model biomarkers. The immunoassay protocol contained primary antibodies immobilized on gold nanoparticles (Au NPs), secondary antibodies conjugated with DNA concatemer from HCR of primer, auxiliary probe, and signal probe labeled with signal molecules (methyleneblue (MB) and ferrocene (Fc)). In the presence of target biomarkers, the sandwich immunocomplex was formed between the primary antibodies and secondary antibodies bioconjugates carrying numerous signal molecules. As a result, two well-resolved reduction peaks, one was at -0.35 V (corresponding to MB) and other was at 0.33 V (corresponding to Fc; both vs SCE), were obtained in differential pulse voltammetry, and peak currents changed were related to the level of biomarkers. Under optimal conditions, the electrochemical immunoassay exhibited a wide linear response range (0.5 pg mL(-1) to 50 ng mL(-1)) and low detection limits (PSA, 0.17 pg mL(-1); AFP, 0.25 pg mL(-1)) (at S/N = 3). In addition, the immunoassay was evaluated by analyzing simulate human serum sample, and the recoveries obtained were within 99.4-107.6% for PSA and 97.9-108.2% for AFP, indicating the immnuoassay could be applied to the simultaneous detection of AFP and PSA in human serum samples.

Functional Micro‐/Nanomaterials for Multiplexed Biodetection

“…[ 121 ] These electrochemical signals could be further amplified by incorporating a DNA hybridization chain reaction (HCR), which improves multiplexed biodetection performance. [ 122 ]…”

Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis

“…HCR application for the analysis of mercury ions, ATP, and proteins has been also reported. Guo et al [106] used α fetoprotein and prostate specific antigen as model biomarkers. In the presence of analytes, immune complexes were formed on the electrode surface.…”

Thioflavin T: A versatile optical probe for chemo and biosensing

“…Considering the significance of mutated DNA in fatal diseases like cancer, sensing of both natural DNA and disease related DNA has been targeted by researchers (Cosnier and Mailley, 2008). Some of the sensitive and efficient platforms developed for detection of nucleic acids are Polymerase Chain Reaction (PCR) (Schaerli et al, 2009;Tassone et al, 2008), DNA microarrays (G. Ventimiglia and S. Petralia, 2013;Sassolas et al, 2008), rolling circle amplification (RCA) (Ji et al, 2012), blotting methods (Glenn and Andreou, 2013;Huang et al, 2010), hybridization chain reaction (HCR) (Guo et al, 2016) and so on. Although these conventional techniques are commonly used, but they involve complex procedures, thus, limiting their utility in wider scenarios.…”